人肝素酶基因正反义荧光真核表达载体的构建及肺癌细胞转染

目的: 构建正义和反义人肝素酶与绿色荧光蛋白真核共表达载体,并分别转染人低转移肺癌细胞株95C及高转移细胞株95D. 方法: 采用基因重组技术,用EcoRⅠ从pcDNA3-Hpa质粒上切下约1.7 kb的人肝素酶全长cDNA片段,然后连入 pIRES2-EGFP质粒的EcoRⅠ酶切位点上,经BamHⅠ酶切鉴定出正义和反义表达载体,并用脂质体法将肝素酶正义真核表达载体转入人低转移肺癌细胞株95C、反义真核表达载体转入人高转移肺癌细胞株95D,G418筛选后,荧光倒置显微镜检测转染结果. 结果: 经BamHⅠ酶切后,正义质粒形成5.3 1.7 kb两条DNA片段,而反义重组质粒为6.5和0.5 kb两条DNA片段,与理论计算值完全一致;转染后肺癌细胞倒置荧光显微下呈绿色荧光. 结论: 成功构建了人肝素酶的正、反义真核表达载体,并成功转染人低、高转移肺癌细胞株,为进一步研究正、反义肝素酶基因转染对肿瘤细胞的影响奠定了基础.

Construction of sense and antisense human heparanase and green fluorescent protein eukaryotic co-expressing vectors and their expression in lung cancer cell lines

AIM: To construct sense and antisense human heparanase and enhanced green fluorescent protein eukaryotic expressing vectors and to transfect these vectors into lung cancer cell lines. METHODS: The human Heparanase cDNA fragment contained in the pcDNA3 Hpa vector was cloned into the enhanced green fluorescent protein eukaryotic expressing vector pIRES2 EGFP in cis direction or trans direction. The recombinant vectors were further identified by digestion of Bam H I and transfected to lung cancer cells by liposome method. After selection with G418, the transfected efficiency was observed under fluorescent inverted microscope. RESULTS: After digested by Bam H I, two fragments with the length of 5.3 and 1.7 kb, respectively, formed in sense fluorescent eukaryotic expressing vector (pIRES2 EGFP sHpa), while two other fragments with the length of 6.5 kb and 0.5 kb, respectively, formed in antisense fluorescent vector (pIRES2 EGFP aHpa). Electrophoretic results were completely identical with the theoretical calculation. Green fluorescence of the transfected cells was observed under fluorescent inverted microscope. CONCLUSION: Human heparanase sense and antisense fluorescent eukaryotic expressing vectors are successfully constructed and transfected to lung cancer cells.